Recent advances in molecular biology are positioning gene and gene-modified cell therapy on the cusp of an extraordinary revolution in patient care for presently unmet medical needs. However, the bioprocessing community is struggling to fulfill growing demands for bio-manufacturing capacity to make current good manufacturing practice (CGMP) viral vectors and particles, and virus-based vaccines.
Cell culture has generated considerable interest in recent years due to the revolution in genetic engineering and biotechnology. Cells are cultured to make proteins, receptors, vaccines, and antibodies for therapy, research, and for diagnostics. Traditionally, cell culture has been operated in a batch mode. In batch operation, the bioreactor is seeded with a small amount of cells and the cells are grown to a higher density. The cells secrete the product of interest and eventually die due to lack of nutrients at which point the culture is harvested. This method has several drawbacks. First, a large fraction of nutrients is wasted in simply growing up cells and are not used directly for making the product; secondly, product formation is often inhibited due to the buildup of toxic metabolic byproducts and lastly, critical nutrients are often depleted leading to low cell densities and consequently lower product yields. It has long been recognized that perfusion culture offers better economics. In this operation, cells are retained in the bioreactor, and the product is continuously removed along with toxic metabolic byproducts. Feed containing nutrients is continually added. This operation is capable of achieving high cell densities and more importantly, the cells can be maintained in a highly productive state for weeks. This achieves much higher yields and reduces the size of the bioreactor necessary or the footprint of the equipment compared to a batch operation, thus reducing costs. In addition, since the harvest is cell free, the initial cell separation step is eliminated, thus simplifying downstream purification steps. Perfusion operations have tremendous potential for growing the large number of cells needed for human cell and genetic therapy applications. The central problem in perfusion culture is how to retain the cells in the bioreactor while removing their desired, secreted product. People have used hollow fiber filtration as the method of choice due to the large surface area provided by the hollow fibers. However, filtration methods require some means to keep the filter from clogging over the required weeks of operation. Cross-flow filters containing hollow fiber membranes are thus typically used. Here a high tangential liquid velocity is used to keep the surface clean. Hollow fiber filters with pore sizes ranging from 10 nm to 1 μm have become the standard of practice for use in perfusion filters.
The traditional upstream manufacturing process for virus production using cell culture consists of a number of batch operations. Viral production entails three steps (1) the growth of “host” mammalian cells in a bioreactor followed by (2) viral production and (3) harvesting of the virus. Commonly these operations are done separately because of the need of different media for the cell growth and viral production phases. Lastly, the harvesting of the virus is commonly done by using depth filters as a separate harvesting step.
Perfusion using an alternating tangential flow system (see, e.g., U.S. Pat. No. 6,544,424, hereby incorporated by reference) offers a significant advantage to batch production of viruses. Alternating tangential flow (ATF) mode has enabled the growth of mammalian cells to a very high density without incurring the shear caused by standard tangential flow equipment which normally results in cell breakage and loss of yield. In U.S. Pat. No. 6,544,424 the ATF system includes two filter elements: a hollow fiber filter element, and a screen filter element. The hollow fiber filter has a membrane with a pore size of 0.2 micron, which allows the harvest of biological substances, such as monoclonal antibodies, with molecular sizes up to 10 nm. However, as is generally known to people working in the art, viral vectors and vaccines, such as retrovirus (e.g., lentivirus), adeno-associated virus (AAV), influenza virus, etc., do not appear in the harvest stream. This size exclusion by the hollow fiber membrane could be due to a combination of aggregation of the viral particles and/or surface interactions with the polyether sulfone hollow fiber membrane. It is known to people proficient in the art that commercially available hollow fiber filter elements having pore sizes of 0.2 μm do not function in an ATF perfusion system for harvesting viruses such as lentivirus (80-120 nm), adeno associated virus (AAV, 20-30 nm) or influenza virus (80-130 nm).
U.S. Pat. No. 6,544,424 also describes a filter element consisting of a screen with pore sizes between 20 μm and 70 μm for perfusion or media exchange of adherent cells using microcarriers. Here the microcarrier is retained by the screen filter element in the bioreactor, due to their relatively large size of 190 microns (GE Healthcare Life Sciences website). The sieve filter cannot be used for producing viruses by perfusion using suspension cells because the suspension cells would flow out of the perfusion filter and be depleted in the bioreactor. The benefit of using a suspension culture compared to a microcarrier based cell culture is that the suspension culture bioreactor and cross-flow filter assembly are more easily scalable. The screen filter element would defeat the purpose and allow passage of both viruses and suspension cells into the permeate and not act as a cell retention device which is its intended purpose.
Unfortunately, a perfusion process which allows both media exchange as well as viral recovery has eluded investigators. Therefore, there is a need for a filter element that would retain cells in suspension as well as allow passage of viral particles into the harvest stream as part of an alternating tangential flow system.